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Dissecting gene expressions in nucleus versus cytoplasm with single-cell resolution

Single-cell ITP for physical fractionation of cytoplasmic versus nuclear nucleic acids (NAs)

Single-cell analyses have become powerful tools to explore the heterogeneity of cell populations such as tumours and developing embryos. Microfluidics have significantly contributed to this field, particularly to reduce the cost and improve the throughput of single-cell RNA sequencing and DNA sequencing.

State-of-the-art technologies have made it practical to obtain sequencing data from thousands of single cells with affordable cost. Existing single-cell sequencing technologies, however, are unable to analyse nuclear versus cytoplasmic RNAs separately and simultaneously as these sub-cellular fractions have nearly identical biochemical properties and easily cross-contaminate. Hence, there remains challenges to discern correlation, localization, and translocation of RNA molecules within a cell at single-cell resolution.

In this blog, we introduce microfluidic approaches leveraging isotachophoresis (ITP) for highly stringent fractionation of NA from the nucleus versus the cytoplasm from single cells.  This enables integrated analyses of subcellular gene expressions in single cells with high-throughput RNA sequencing. ITP is an emerging alternative to traditional sample preparation of NA from complex biological samples.1-5 It leverages the high electrophoretic mobility of NA relative to impurities and inhibitors to extract, purify, and deliver DNA and/or RNA to a downstream assay.6

ITP can focus NA at the interface between a low mobility trailing (TE) and high mobility leading electrolytes (LE) under an electric field. It offers highly sensitive,7-13 robust,14 rapid,5,15 and extremely selective16-20 sample preparation. Focusing reactant biomolecules (e.g. probe and target) into the same ITP zone has also been shown to dramatically increase reactions rates and achieve rapid assays.21 For example, ITP mix-and-react assays have been used to increase DNA hybridization reactions by up to 14,000 fold in homogenous reactions.11,15,22  It has also been used to control and speed up multiplexed heterogenous reactions between target DNA and DNA arrays11 and between target molecules and probes on fluorescent beads.10

Our approach for the stringent fractionation of NA from single cells hydrodynamically traps and isolates a single cell in a microchannel. The chip then selectively and electrically lyses the plasma membrane while retaining the nucleus membrane relatively intact.  Within 1 second, we initiate electric-field-based extraction of cytoplasmic NA from the lysed cell via ITP.  This physically separates cytoplasmic RNA from the nucleus, and “packs” this aliquot into an ITP zone which is diverted to a dedicated output reservoir.

At the first output, cytoplasmic NA can be recovered for analysis (e.g. for off-chip cytoplasmic RNA sequencing).  Subsequent to this, the cell nucleus is diverted to a second output reservoir on the chip using pressure driven flow.  This in turn is also recovered for off-chip analysis of nuclear NA. Cell trapping, selective lysing, ctyplasmic NA extraction, and nucleus extraction are completed within 5 min. We refer the readers to detailed descriptions of a single-cell ITP protocol and chip, including a narrated video description reported by Kuriyama et al.23

Capturing cell-to-cell variation leveraging single-cell ITP

The single-cell ITP microfluidic approach is robust and sensitive to capture cell-to-cell variations, including cell cycle.24 The method allows simultaneous quantification and correlation analyses of cytoplasmic RNA and genomic DNA from a same single cell via on-chip fluorescence measurement.

Unlike optical fractionation, (fluorescence activated cell sorters) this method provides physically fractionated cytoplasmic contents versus nuclei to downstream assays. The extracted cytoplasmic RNA and nuclei including genomic DNA and nuclear RNA are compatible with reverse transcription and polymerase chain reactions.23,25 We have demonstrated the integration of the method with parallel nuclear and cytoplasmic RNA-seq of the same single cells; and we term this approach single-cell integrated nuclear and cytoplasmic RNA-seq (SINC-seq).26

Leveraging SINC-seq, we explored the landscape of the correlation of gene expressions between nuclear RNA and cytoplasmic RNA with K562 leukemic cells and discovered distinct natures of correlation among cytoplasmic RNA and nuclear RNA which reflect transient physiological states of single cells. In particular, we found the following: cell-cycle-related genes displayed a highly correlated expression pattern between nuclear-RNA-to-cytoplasmic RNA with minor but notable differences; and RNA splicing genes showed lower correlation, suggesting a retained intron may be implicated in inhibited mRNA transport.

Further, a chemical perturbation, sodium butyrate treatment, transiently distorted the correlation along human leukemic cells differentiating to erythroid cells. These data uniquely provided insights into the post-transcriptional regulatory network of mRNA from nucleus toward cytoplasm at the single cell level. At the same time, our study suggests a compelling caution to an approach that approximates the transcriptomic profile of the whole cell with that of a subcellular compartment without careful validation.

What’s next?

There remains further work to develop single-cell ITP as a high-throughput and convenient benchtop tool. We expect that one of the challenges may be addressed by coupling the method with cellular indexing via molecular barcodes. Another substantial challenge is to automate further and remove manual steps including dispensing buffers/samples into and out of the microfluidic system, and such automation may benefit from robotic actuation.

Juan Santiago

 

Juan Santiago is a professor at Stanford University who has spoken previously at the 4Bio Summit: USA.

 

 

Have you seen the agenda yet for the 4Bio Summit: Europe? View it here.

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